Progress on the development of a single line of sight x-ray framing camera

High-speed micro-strip micro-channel plate (MCP) x-ray framing cameras are a well established diagnostic for laser plasma experiments. Each frame acquired with these devices requires a separate image, and with most reasonable x-ray optics, a separate line of sight, causing potential parallax problems. Gated image tubes have a single line of sight capability, but the conventional designs have not been effectively extended to the short gating times of the micro-strip-line MCP camera. A hybrid camera combining image tube and micro-strip-line MCP technology has been under development at LLNL in collaboration with UR/LLE, and KENTECH Instruments. The key feature of this single line of sight (SLOS) hybrid image tube is a deflection assembly that continuously divides the electrons from a single photocathode x-ray image into a set of four electron images. Temporal gating of these images is carried out using a microstripline microchannel plate framing camera module positioned at the image plane of the electron tube. Characterization measurements performed using both X-rays from a Manson source and from laser generated plasmas, will be presented. Some implementation improvements will be discussed. The results will be compared to simulations carried out using the charged particle optics code SIMION. Various dissector designs were simulated in an effort to improve the image quality of the system

Advanced optical imaging in living embryos

Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis

Design and implementation of high magnification framing camera for NIF "ARIANE Light"

Abstract not provide

Design and implementation of Dilation X-ray Imager for NIF "DIXI"

Abstract not provide

A Bayesian method for inferring quantitative information from FRET data

<p>Abstract</p> <p>Background</p> <p>Understanding biological networks requires identifying their elementary protein interactions and establishing the timing and strength of those interactions. Fluorescence microscopy and Förster resonance energy transfer (FRET) have the potential to reveal such information because they allow molecular interactions to be monitored in living cells, but it is unclear how best to analyze FRET data. Existing techniques differ in assumptions, manipulations of data and the quantities they derive. To address this variation, we have developed a versatile Bayesian analysis based on clear assumptions and systematic statistics.</p> <p>Results</p> <p>Our algorithm infers values of the FRET efficiency and dissociation constant, <it>K_d</it>, between a pair of fluorescently tagged proteins. It gives a posterior probability distribution for these parameters, conveying more extensive information than single-value estimates can. The width and shape of the distribution reflects the reliability of the estimate and we used simulated data to determine how measurement noise, data quantity and fluorophore concentrations affect the inference. We are able to show why varying concentrations of donors and acceptors is necessary for estimating <it>K_d</it>. We further demonstrate that the inference improves if additional knowledge is available, for example of the FRET efficiency, which could be obtained from separate fluorescence lifetime measurements.</p> <p>Conclusions</p> <p>We present a general, systematic approach for extracting quantitative information on molecular interactions from FRET data. Our method yields both an estimate of the dissociation constant and the uncertainty associated with that estimate. The information produced by our algorithm can help design optimal experiments and is fundamental for developing mathematical models of biochemical networks.</p

Spectroscopic infrared scanning near-field optical microscopy (IR-SNOM)

Scanning near-field optical microscopy (SNOM or NSOM) is the technique with the highest lateral optical resolution available today, while infrared (IR) spectroscopy has a high chemical specificity. Combining SNOM with a tunable IR source produces a unique tool, IR-SNOM, capable of imaging distributions of chemical species with a 100 nm spatial resolution. We present in this paper boron nitride (BN) thin film images, where IR-SNOM shows the distribution of hexagonal and cubic phases within the sample. Exciting potential applications in biophysics and medical sciences are illustrated with SNOM images of the distribution of different chemical species within cells. We present in this article images with resolutions of the order of λ/60 with SNOM working with infrared light. With our SNOM setup, we routinely get optical resolutions between 50 and 150 nm, regardless of the wavelength of the light used to illuminate the sample

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Forster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.Peer reviewe